Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents

ABSTRACT

The detection of microorganisms concentrated from body fluids as serum, sputum, pericardiac fluid, urine or other fluid on the surface of retaining membranes by specific partners of reaction that bind to antigenic components of the retained organisms is facilitated when the organisms in suspension in body fluids and/or concentrated on the membrane are treated with a high molar concentration of a chaotropic agent as guanidine, urea, isothiocyanate, thiourea.

FIELD OF THE INVENTION

[0001] The present invention is in the field of diagnostic and isrelated to a method and kit for the rapid detection of wholemicroorganisms on retaining membranes by the use of chaotropic agents.

BACKGROUND OF THE INVENTION

[0002] Detection of microorganisms in body fluids, water and alimentaryproducts allows diagnostic and prognostic of many infectious diseases.Cells and microorganisms needed for the making of vaccines demands asevere control of the sterility of the produced media, usually achievedby culturing an aliquot of the medium whose sterility one wants tocontrol, in a growth-promoting medium during several days. Said growthand identification of microorganisms are time consuming, difficult andfrequently takes two to three weeks before results are available.

[0003] For diagnostic purposes, such a method of determination relyingon culture of blood sample or of sputum exists, when there is a need todiagnose the presence of mycobacterial entities. The time needed toarrive at a diagnostic conclusion is however very long, extendingsometimes to 4 weeks. For other microorganisms, such as the etiologicalagents of malaria (especially Plasmodium falciparum, leishmaniasis,gonorrhea, syphilis, tuberculosis, malaria, tick-borne Lyme disease, aswell as meningitis due to a variety of vectors such as Neisseria,Haemophilus, Streptococcus, Listeria and Mycobacterium, a rapiddiagnostic method putting the whole pathogen in evidence does not existand there is a need for such rapid method.

[0004] Rapid methods of detection of antigens and antibodies are nowcurrently in use for antibodies and proteinic and glycoproteinicantigens whose size is sufficiently small for them to be transported ina lateral flow chromatographic system. For larger entities (such aswhole bacteria and eukaryotic organisms), this technique is inapplicablebecause the analytes are too big to move with the flow. Thesemicroorganisms of large size are also able to condense on retainingmembranes in such a way that the sensitivity of their detection in situis considerably enhanced.

[0005] However, an intolerably high level of false positive or negativeresults is also observed. The phenomenon of immunological quenchinginduced by body fluids is well known. This quenching prevents therecognition of the whole organisms by specific binding partners asantibodies and induces false negative results.

STATE OF THE ART

[0006] The document EP-0306206 describes a diagnostic device comprisinga funnel that channels the fluid under analysis onto a defined part of afilter membrane that retains the analyte. The funnel is thereafterremoved and the membrane is further treated by signal-generatingmaterials and other reagents as washing fluids, applied simultaneouslyto the test area and adjacent area of the membrane. The simultaneoustreatment of adjacent area supposedly provides a negative control toassist in detecting non-specific binding and thereby false-positiveresults. In said document, the sample is confined to a discrete area ofthe filter and the whole of the filter is then treated with reagent,thereby giving a true negative control. This document mentions water asfluid passing through the restricted aperture and omits to describe thetype of membranes used. Such an improved device relying on directdetermination of the presence of pathogens on the membrane is indeedapplicable only with water. More charged fluids such as serum,accumulated into a single discrete area of the filter tend to clog thisfilter up, prevent the easy flow-through of subsequently added fluids(such as a wash medium) and cause the succeeding washing fluids, appliedto the whole of the membrane, to flow around that area withoutpenetrating it. Further, even with water, the device is specificallydeveloped and meant to visualise false positive cases, therebyindicating that the occurrence of false positive cases is possible, evenwith water. No such device applied to detect. whole microorganisms bydirect labelling is in use for body fluids such as serum, cerebrospinalfluid, pericardial fluid, urine or other body fluid.

[0007] The use of a chaotropic agent like sodium thiocyanate has beenadvocated for suspensions of microorganisms in water before theircondensing on a membrane filter, but the concentration advocated is 0.04molar and only water is recognised as a fluid suitable for treatment(document JP-05034350). This teaching addresses the issue of detectionof microorganisms in water, whose clumping may be prevented ordeliquesced into singly suspended organisms with the help of lowconcentrations of disrupting agents.

[0008] The increase in sensitivity of an immunochemical assay ofcollagen has been claimed in the presence of a chaotropic agent. Neitherurea, nor guanidine are mentioned as suitable, but thiocyanate used at aconcentration of 0.1 to 1.6 molar included in the reaction mix is(document WO92/16846). This teaching addresses the issue of collagensolubilised in an assay medium containing a chaotropic agent asisocyanate and recognised by a binding partner.

[0009] Similarly, a treatment of a sample with various detergents and achaotropic agent as urea prior to immunoassay has been claimed to beapplicable to blood samples investigated by immunoassay for the presenceof viruses as hepatitis C virus (document WO99/06836). In this case, thechaotropic agent is contacted together with different surfactants withthe sample under analysis, not as a washing solution, and no attempt ismade to detect in a direct way the pathogenic entity condensed on thesurface of a membrane, but a lateral flow immunoassay is used in thedetection of the analyte present in the treated biological fluid.

[0010] The document U.S. Pat. No. 5,714,343 describes a deviceconsisting in absorbing pads surmounted by a retaining membrane. Thefluid is passed through the retaining membrane and the microorganismspotentially retained on the membrane are visualised by a chromogenicagent having an oxidation potential such that the reagent can be reducedby microbial dehydrogenase, yielding a visibly coloured productindicative of the presence of microorganisms in the sample. Theretaining membrane having pores (0.75 to 1.2 μm) larger than thedehydrogenase-active microorganisms it is supposed to retain, thecapture of the bacteria on such filters is achieved by mechanicalretention. The reason for this unlogically great pore-size used at therisk to let a large amount of the analyte pass through the filter, isthe possible occurrence of false positive cases with smaller pores,since other reducing compounds than bacteria (e.g. free reducingenzymes, ascorbic acid, glutathione) may then,be retained, yieldingfalse positive results. Further, although the device is said to beuseful for water, blood, milk and urine analysis, only physiologicalwater is described in examples 1 to 7. The principal advantage of thismethod is the signal-generating material, consisting in a chromogen thatforms a visible precipitate of formozane after reaction with bacterialdehydrogenase. Growth of the bacteria in vitro for several days aftercollection on the retaining membrane is therewith avoided, and much timesaved. Many bacteria and yeasts as well as leukocytes possess adehydrogenase activity and the detecting method does not discriminatebetween them, except for the selective destruction of dehydrogenasesynthesised by gram positive bacteria, leaving the dehydrogenase fromgram-negative bacteria still active. This selective destruction ofdehydrogenase produced by gram-positive bacteria is achieved by eitherincubation of the sample with octyl glucoside, which suppresses thedehydrogenase activity of all gram+ bacteria tested, or else 0.5 Mguanidine, that suppresses the dehydrogenase activity of only some gram+bacteria while at least one gram-bacterium, Pseudomonas, is alsoaffected (only living bacteria possessing active dehydrogenase).

[0011] Therefore, one of the drawbacks of said technique is that it doesnot allow a specific detection, because a large number of microorganismspossessing active dehydrogenase and other reducting enzymes are detectedand identified.

[0012] The documents U.S. Pat. Nos. 4,695,537 and 4,808,518 underlinethe fact that hypertonic solutions of agents as sodium chloride,potassium thiocyanate, guanidine and others may be applied to antigensin suspension and to intracellularly located antigens, at concentrationsbetween 1.0 and 5.0 M, preferably between 2.0 and 3.5 M. Theseconcentrations do not reduce the antigenic properties of the antigen butimprove the yield.

[0013] The document EP 0 234 941 indicates that treatment of viralsubunits with denaturing concentrations of guanidine (from 5 to 8 molar)(after the purification of the virus and its disruption into subunitswith detergents) improves the purity of these subunits for theadsorption of antibodies in an Elisa. It is known that denaturingconcentrations of chaotropic agents destroy protein assemblies intosubunits, as is the case with the alpha and beta subunits of humanchorionic gonadotropin. Sometimes, as is the case with the subunits ofHCG, the immunogenicity of the subunits is not affected by thishypertonic treatment. Tuberculoproteins are isolated by treatingtubercular material with chaotropic agents (guanidine, urea and phenol)which reduce the tubercle pathogen to immunogenic tuberculoproteins(FR-2082226). However, these documents do not suggest the maintenance ofthe pathogen under analysis whole and intact, without any reduction intosubunits, so that it remains of a size sufficient to be mechanicallyretained on a filtering membrane and thereafter its direct detection.

[0014] Aims of the Invention

[0015] The present invention is related to a method and kit for therapid detection of whole microorganisms, which do not present or reducethe possible false positive or false negative results affecting themethod of the state of the art.

SUMMARY OF THE INVENTION

[0016] A simple detection system able to demonstrate in a direct way, ina sensitive way and in a specific way the presence of whole pathogensbelonging to the bacterial kingdom or else eukaryotic parasites asPlasmodium falciparum or toxoplasma in various body fluids as serum,cerebrospinal fluid, pericardial fluid etc. condensed on a retainingmembrane, is not yet available, although such a detection would greatlyimprove the rapid diagnostic of several pathogens. Such a system must,per force, exploit the specific binding capacities existing amongspecific binding partners for the microorganisms one wishes to detect,as specific antibodies, protein A, protein G.

[0017] The occurrence of false positive results are due to contaminatingsubstances present in the body fluids wherein the microorganisms aresuspended and quenching induced by them, which adsorbs to the surface ofthese microorganisms and prevent their recognition by specific bindingpartners (either when these organisms are in suspension in these bodyfluids or after, these microorganisms have been condensed out of thesefluids, on the surface of retaining membranes).

[0018] The present invention is related to a method for the detection ofone or more microorganisms present in a liquid sample, preferably abiological liquid sample, said method comprising at least the followingsteps:

[0019] (a) possibly adding to said liquid sample a reagent solutioncomprising a chaotropic substance having preferably a concentration ableto rupture hydrogen bonds;

[0020] (b) filtering the liquid sample through a filter having a poresize which is small enough to prevent passage of microorganisms throughthe filter, but large enough to permit passage of any soluble materialpresent in the sample, whereby the microorganisms are retained on thefilter;

[0021] (c) possibly passing said reagent solution comprising achaotropic substance through the filter having the retainedmicroorganisms thereon, said chaotropic agent solution having preferablya concentration able to rupture (suppress) hydrogen bonds, and

[0022] (d) passing one or several labelled reagents (signal-generatingreagents) through the filter, at least one of said reagents beingspecific for the microorganisms to be detected.

[0023] The chactropic agent solution comprises urea, guanidine,thiourea, isothiocyanate or a mixture thereof and the concentration ofthe chaotropic agent in the solution is higher than 2M, preferablybetween 4 and 8M (denaturating concentration).

[0024] The microorganisms could be present in any biological sample,preferably in a charged biological sample such as sputum, blood, serum,plasma, cephalo-rachidian fluid or urine obtained from any animalpatient, including the human and are preferably pathogenic bacteria oreukaryotic parasites detected either simultaneously or successively.

[0025] Contaminants of beverages and foods, as listeria, yeasts andmolds are also detectable. The type of filter used to retain theanalytes under study is usually cellulose, paper, nitrocellulose ornylon, or other types selected by the man skilled in the art.

[0026] The specific labelled reagents used for the detection ofmicroorganisms are preferably antibodies, possibly coupled directly orindirectly to a marker, such as gold micellae, biotine, enzymes,chromophores, stained latex beads, enzymes, fluorophores, radioactivecompounds or a mixture thereof.

[0027] The present invention is related also to a kit comprising meansand media for performing the detection method according to theinvention, said kit comprising filtering membranes of predetermined poresize to retain specific microorganisms, one or several chaotropicagents, one or several labelled reagents (signal-generating reagents),one of them being specific for the monitored microorganisms.

[0028] If necessary, the method and kit could be adapted to allow therapid detection of microorganisms present in a sample by using anautomate and means for performing automatically the specific detectionaccording to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The presence of microorganisms as the Koch's bacillus(Mycobacterium tuberculosis), Plasmodium falciparum, or other bacteriaand parasites difficult to diagnose, as leishmaniasis, gonorrhea,syphilis, tick-borne Lyme disease, as well as meningitis due to avariety of vectors such as Neisseria, Haemophilus, Streptococcus, andListeria, and yeasts and worms, is detected in human and animal serumand other body fluids by condensing the microorganisms present in thesefluids on the surface of a membrane that allows the passage of thefluid, but retains the microorganisms on its surface. Thereafter bywashing the membrane to eliminate contaminants, treating the membranewith a chaotropic agent as guanidine, urea, guanidine isothiocyanate orsimilar agent, at a concentration high enough to clean themicroorganisms of all the quenching elements that prevent theirrecognition by specific binding partners, treat the retaining membraneand the microorganisms condensed on its surface with a binding partnerspecific for the microorganism, such as antibodies, thereafter onewashes the membrane free of excess binding reagent and label theantibodies bound to the microorganism with a binding partner specificfor the class of gammaglobulins of the animal species in whichantibodies against the microorganism were raised, e.g. rabbit, goat,donkey, as antibodies against rabbit or goat or donkey gammaglobulins,protein A or protein Gj, said binding partner being itself coupled to amarker (i.e. an agent susceptible to amplify the labelling, as colloidalgold or end enzyme).

[0030] The detection of microorganisms having the size of bacteria orbigger, (spirochetes, trypanosomes and worms) present-in body fluids asserum and urine obtained by means of specific binding partners asantibodies, protein A or protein G is not easy.

[0031] However microorganisms in solution or condensed on the surface ofa membrane resist the disruptive action of high molar concentrations ofchaotropic agents as guanidine, urea, thiocyanate, thiourea,isothiocyanate, perchlorate etc. while becoming readily accessibletherewith to recognition by specific binding partners. The samemicroorganisms condensed from the same sera on the same retainingmembranes and treated with the same chaotropic agents used at a molaritytoo low to exploit their. disruptive potential on proteins (as forexample 0.5 molar guanidine used to destroy the activity of gram+bacterial dehydrogenase) are not recognised by their specific bindingpartners. Therefore a tenfold higher concentration of the chaotropicagents is unexpectedly beneficial in the conduct of an immunoassay.

[0032] Molarities of urea and guanidine ranging from about 4 to 8 areneeded to have an effect. With other chaotropic agents, as thiourea andisocyanate, lower concentrations ranging between 2 and 5 molar, areadequate.

[0033] High molar concentrations of chaotropic agents as guanidine andurea have been for a long time applied, mostly together with organicsolvents as phenol and chloroform, to liberate the nucleic acids DNA andRNA from eukaryotic cells, from bacterial cells as Escherichia coli andfrom virus entities. The fact that nucleic acid is obtained followingtreatment of cells and viruses with chaotropic agents indicates that thecell membranes, the nuclear membranes and tertiary structures ofproteins in viruses are destroyed by this treatment. This effectprecludes any evident application of chaotropic agents at high molarconcentrations for the purpose of the present invention.

[0034] Chaotropic agents as urea and guanidine applied at concentrationsranging between 4 to 8 molar, useful and necessary for the exploitationof their protein denaturing properties and their ability to suppresshydrogen bonds, are currently used either to eliminate proteins fromnucleic acid preparations or else to separate protein subunits,thereafter isolated by separation methods based on the differentphysico-chemical properties of the separated products. For example, thealpha and beta subunits of chorionic gonadotropin are separated in thepresence of 8 molar urea at 40° C. and the two subunits are subsequentlyisolated by passage on a DEAE-cellulose chromatography column. Purifiedtuberculoproteins are isolated by submitting tubercular material to aprecipitation stage at an acid pH, carried out in the presence of asubstance capable of rupturing hydrogen bonds, as urea, guanidine,formamide, phenol, etc. The precipitated material is thereaftercontacted with a modified cellulose bearing a basic group such as DEAE(see document FR-2082226). In these cases, the molarity of thechaotropic substance is sufficiently high to exploit its disruptingproperties on substances kept in solution, with the purpose toeffectuate a separation and solubilisation of the various substancescontained within the original preparation.

[0035] The main problem encountered in the development of assays aimingat detecting in a direct way pathogens condensed on a retaining membraneis the quenching induced by charged liquid media as serum, quenching ofsuch a nature that the microorganism is not anymore readily recognisedby specific binding partners. Many sera induce a quenching thatconsiderably reduces the signal that is detected after application ofsignal-producing elements. More particularly, the pathogen isolated fromsome sera and condensed at the surface of the retaining membrane doesnot react to antibodies directed against it, which precludes thepossibility to thereafter visualise the presence of these antibodies bysignal-amplifying binding partners as gold-labelled protein A or asperoxidase-labelled antibodies specifically binding with gammaglobulins.Whereas some sera and less charged fluids as urine, culture media andwater are satisfactory in this sense, the fact that some sera inducefalse-negative results precludes the routine use of such methods for thedetermination of the presence of pathogens.

[0036] Quite unexpectedly, it was discovered that high molarconcentrations of chaotropic agents as guanidine, urea, thiourea,isocyanate, applied directly on the liquid sample under analysis and/oron the retaining membrane following the condensing of the microorganismon its surface, greatly facilitates the reaction of specific bindingpartners with the microorganism under analysis. A device consisting in acartridge containing absorbent pads, topped with a retaining membrane,is currently available for the making of such assays as described in thedocument U.S. Pat. No. 5,714,343 incorporated herein by reference. Usingsuch a device, the following experiments were done, which may serve asexamples.

EXAMPLE 1

[0037] 500 μl of human serum spiked with tuberculous bacillus was passedover a membrane whose mean pore size was 0.45 microns. This pore size issmall enough to retain the great majority of the bacilli, whose meansize is 0.6×4 μm. After passage of the liquid sample, the retainingmembrane was washed with 200 μl of washing medium. The membrane wasfurther washed with 200 μl of a 6 molar solution of guanidinehydrochloride at pH 8.0. A 200 μl wash with washing medium eliminatedthe quanidine and the membrane was thereafter treated with 100 μl of adilute solution of rabbit antibodies against mycobacterium tuberculosis.The antibodies were obtained in rabbits repeatedly inoculated withheat-killed Mycobacterium tuberculosis (Difco). After a 200 μl wash,100. μl of gold-labelled protein A at a concentration of A₅₂₀=0.75 wereapplied, with a positive result consisting in a red-pink spot in themiddle of the membrane.

[0038] The same analysis performed in the absence of a chaotropicreagent produced a diffuse barely coloured spot instead.

[0039] The same analysis performed with a solution of guanidine whoseconcentration was lowered to about 4.5 moles yielded a spot whoseintensity was similar to the positive control (6 molar guanidine).

[0040] The same analysis performed with a solution of guanidine whoseconcentration was increased to 7 moles yielded a spot whose intensitywas similar to that obtained with about 6 molar concentration inguanidine.

EXAMPLE 2

[0041] An analysis identical to that described in Example 1 wasperformed with a solution that was 7 molar in urea. The same positiveresult as in example 1 was obtained, whereas concentrations lower thanabout 4.5 molar were ineffective in revealing a satisfactory signal. Aconcentration of urea superior to about 8 molar reduced the signal.

EXAMPLE 3

[0042] The same experiment as in Example 1 was conducted, with theguanidine wash solution brought to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,10.0, 11.0, 12.0 and 13.0 with concentrated HCl or 10 molar NaOH. Thesolutions of the chaotropic agent that were of an acidity equal orinferior to pH 6.0 were much less satisfactory that those whose pH wasabove neutrality. At a basic pH 13.0, the signal was reduced. Optimalsignal delivery was obtained with a solution of guanidine at pH 9.0.

EXAMPLE 4

[0043] The same experiment as in example 1 was conducted with thechaotropic agents 4 molar potassium thiocyanate, 2 molar thiourea, 6molar guanidine containing 2 molar thiourea, 3 molar urea mixed with 3molar guanidine. Solubilisation difficulties due to the presence ofvarious surfactants currently used in immunoassays and known from theman of the art did not allow higher molarities, concentrations forthiocyanate and thiourea than those here applied. These chaotropicagents were not superior to urea or quanidine used alone.

EXAMPLE 5

[0044] These experiments showed that, when no sputum is used, a membranewith a pore size 0.45 μm has a satisfactory retention power. However,when sputum is used, the 0.45 μm pore sized membrane tends to clog upeasier. A membrane with a larger pore size is preferable. To furtherfavour flow, a glass fiber pre-filter is needed, that retainsundissolved matter and viscous matter present in sputum.

[0045] Sputum from a TB tuberculosis patient was collected at thePneumology department of the University Hospital of Strasbourg (France).The sputum underwent regular analysis and was found to contain TBbacilli by the bacilloscopy technique based on the Ziehl-Nielsen Stain.The sputum was fluidised by addition of 0.25% N-acetyl-cysteine and 1%NaOH (final concentrations). This fluidising method is standard inmycobacteriology for the treatment of sputum before its use in cultures.The NaOH is added as a fluidisation medium and also in order todecontaminate the sample and kill all other bacteria present, beforeculture for tubercle bacilli is initiated. The NaOH is not indispensablefor the liquefaction and N-acetyl-cysteine, at concentrations between0.5% and 2% and alkaline pH, is able to liquefy sputum in two minutes.Other methods of fluidisation, based on hypochlorite or sodium dodecylsulphate, were found to work just as well. In particular, bleach may beappropriate for low income countries because it is cheap, availablelocally and it better respects the integrity of the retaining membranethan NaOH. An alteration of the retaining membrane, made ofnitro-cellulose, was observed when the sample fluid was treated withNaOH at 1%. A neutralisation of the fluid with HCl was needed beforepassing it on a nitro-cellulose retaining membrane. Other means offluidisation (bleach, dithiothreitol) proved compatible with theretaining membranes used. Any fluidising agent that respects theantigenicity of the bacteria and does not alter the retaining membraneis satisfactory.

[0046] A fiber-glass prefilter pad was placed on top of thenitrocellulose retaining membrane, so as to retain the unsolubilisedmaterial remaining in the fluidised sputum. 200 μl of fluidised sputumwere passed through the prefilter and the 0.8 μnitrocellulose membrane,before the fiberglass prefilter was removed. The retaining membrane waswashed with 2 times 100 μl of 0.2M phosphate pH 7.2 containing 0.2%Tween 20. After the wash, the membrane was treated with 6 M guanidine,and further contacted with a rabbit antiserum against M. tuberculosisappropriately diluted 1:160 in phosphate-buffered saline pH 7.2,containing 0.2% Tween 20. After a washing step, the signal present onthe membrane amplified with a reagent consisting in 30 nmgold-sensitised rabbit anti-goat IgG. The sensitivity of the systemdetects about 3000 mycobacterial entities/ml.

EXAMPLE 6

[0047] The detection of mycobacteria in sputum was done as in example 5,with a membrane whose pore size was 0.45 μm. This small pore size wasfound acceptable although it retarded the flow rate, but a slower flowof the reagents enhanced the sensitivity of the method by allowing alonger contact and prolonged reaction time of the reagents with theantigens. Rabbit anti-mycobacterial serum was used to label the antigenretained on the membrane. The secondary partner of reaction was composedof antibodies against rabbit IgG raised in donkeys. The enhancer was 30nm-gold-labelled Protein G., which reacts well with equine antibodies.The sensitivity reached was about 3000 entities/ml.

EXAMPLE 7

[0048] A membrane with a pore-size of 0.45 μm was used to collect themycobacteria suspended in sputum in an experiment applied as in example5. After labelling the mycobacteria present on the membrane withspecific rabbit antibodies, a secondary binding partner consisting ofProtein A-20 nm colloidal gold, was applied (Sigma Chemical Co, StLouis, Mo). The results were similar to those obtained in example 6,when the secondary antibody was gold-sensitised goat anti-rabbit IgG,i.e. 3000 particles/ml.

EXAMPLE 8

[0049] Human infections caused by Chlamydia trachomatis primarilyinvolve the eyes and the genital tract. The morphology of the chlamydiaeresolves into Elementary Bodies (0.2 to 0.4 μm) that can be detected byimmunofluorescence or enzyme linked immunosorbent assays and ReticulateBodies which is the intracellular, metabolically active form (0.6 to 1.0μm), rarely observed microscopically The laboratory diagnosis is basedon a variety of methods (complement fixation, microimmunofluorescence,enzyme linked immunosorbent assays, direct microscopic examination,isolation of the microorganisms and nucleic acid techniques). Forgenital tract infections, specimens for assay are urethral and cervicalswabs, and urine, with the aim to detect the etiological agent.

[0050] Human urine was spiked with decreasing amounts of the pathogenChlamydia trachomatis Elementary bodies. Antibodies raised in rabbitswere used to label the pathogen. One and a half millilitres of thespiked urine were passed on a filter with mean more size 0.2 μm, thatwas able to retain the pathogen on its surface. No prefilter was useful.Revelation of the presence of the pathogen was done by treating themembrane first with 5 M guanidine at pH 8.0, thereafter with thespecific antibodies, followed with Gold-protein A, that reacts withrabbit gammaglobulins. In a second analysis, antibodies raised in goatswere used. Gold-labelled protein G was used to reveal the presence ofthe goat antibodies and an enhancer consisting in Gold-labelled piggammaglobulins was finally applied. The sensitivity of this detectionsystem was compared to that obtained by the rapid test chlamydia, acommercial diagnostic kit (Abbott). Whereas a single amplificationsystem (rabbit antibodies and Gold-protein A) yielded similar results,the use of an enhancer (Gold-labelled pig antibodies) increased thesensitivity two-fold.

EXAMPLE 9

[0051] Toxoplasmosis is caused by Toxoplasma gondii, a sporozoan whoseindividual cells are 4 to 7 μm long. Diagnosis relies essentially ondetection of specific antibodies in serum but the sites most commonlyattacked are the lymph nodes, brain, eyes and lungs. Direct examinationof sputum, vaginal exudates, spinal, pleural and peritoneal fluids arepossible, but rarely practised. No diagnostic kits exist for directexamination of sputum and body fluids.

[0052]Toxoplasma gondii organisms were mixed with sputum, the sputum wasliquefied and 1 ml passed on an 0.8 μm membrane fitted with a prefilter.The same procedure as in example 7 was followed. The results wereexcellent, with the detection of about 200 pathogenic entities in thesample.

EXAMPLE 10

[0053] Few diagnostic kits for the detection of Neisseria gonorrhoeaeare available. The laboratory diagnosis of gonococcal infection is basedprimarily on the identification of the etiologic agent by microscopicexamination and by culture. Cultures derived from sterile sites(cerebrospinal fluid, blood, synovial fluid) usually provide adefinitive diagnostic but positive cultures from non-sterile sites areof uncertain value. No serologic test is commercially available yet.

[0054] Sputum was spiked with known concentrations of Neisseriagonorrhoeae antigen, as in example 5. The pore-size of the retainingmembrane was 0.45 μm, compatible with the size of the organisms (0.6 to1.0 μin diameter). The solubilisation of the sputum was done with 0.2%Sodium dodecyl sulphate at pH 9.5, treatment that was found not to alterthe immunoreactivity of the pathogen. The presence of N.gonorrhoeae-specific rabbit IgG on the surface of the membrane,indicative of the presence of the pathogen, was further pursued as perexample 7, with similar results.

EXAMPLE 11

[0055] The microscopic observation of the adult schizonts of Plasmodiumfalciparum in blood smears is the laboratory diagnostic of malaria.

[0056] Human fresh whole blood was spiked with formalin-inactivatedschizonts. After the spiking, the blood was hemolysed with 1% Nonidet P40 (final concentration) and 500 μl of the fluid was passed on a glassprefilter and collected on a retaining membrane with pore size 0.8 μm.Guanidine at a 4 molar concentration at pH 8.0 was found satisfactory inthis analysis. A high concentration of 8 molar urea was also foundadequate but this remarkable resistance of the schizonts may have beenobtained by their fixation with formalin. The mouse antibodies used tolabel the antigen were revealed with Gold-labelled Protein G. Distinctred spots on the membrane signalled the presence of individualorganisms.

[0057] A great number of possibilities were investigated, using eitherantibodies labelled with peroxidase or with gold, using protein A andprotein G labelled with gold or with peroxidase, and the usefulness ofsecondary amplification steps was also investigated. The most reliableand easy method was found to be the one described in example 7: 500 μlto 1.5 ml (depending on the proteinic load of the processed sample) ofsample is passed through a retaining membrane. The organisms presumablyconcentrated on the surface of the membrane are then treated with asolution 6 molar in guanidine hydrochloride at pH 8. After a wash, themembrane is treated with rabbit antibodies against the analyte and thepresence of the antibodies potentially attached to the antigenicdeterminants of the analyte are put in evidence with gold-labelledprotein A.

EXAMPLE 12

[0058] A treatment of the analysed sample directly with highconcentrations of a chaotropic agent, before the concentration of theanalyte on the retaining surface of a filtering membrane, possiblyfollowed by a washing with the chaotropic agent, yields the bestresults.

[0059] 100 μl of sputum found positive for TB by a microscopicexamination were solubilised with 100 μl of solubilising solutionconsisting of 5% N-acetylcysteine and 0.5% mercaptoethanol in 1% NaOHbrought at pH 12. This solubilising solution is standard procedure butother solubilising methods (e.g. sodium hyposulphite) known by theperson skilled in the art are equally applicable.

[0060] After the sputum has been digested (5 minutes at roomtemperature), the sample is mixed with 1.3 ml of a solution 7 molar inguanidine at pH 8.5 containing 0.01% Tween 20, and processed as perexample 7.

[0061] A red central spot is observed after completion of the test, notobservable when negative samples are processed in an identical manner.

EXAMPLE 13

[0062] 100 μl of sputum found positive for TB by a microscopic analysiswas mixed with 100 μl of a solution that was 7 molar in guanidine at pH12.00. After solubilisation of the sputum, 100 μl of NaClO at 12° wasadded to the mixture and digestion of the sputum was pursued during 15minutes at Room Temperature. The sample was thereafter mixed with 1.3 mlof a solution that was 7 molar in guanidine at pH 8.5 and processed asper example 12.

[0063] A red central spot is observed after completion of the test, notobservable when negative samples are processed in an identical manner.

1. A method for the detection of one or several microorganisms presentin a liquid sample, preferably a biological liquid sample, comprisingthe following steps of: (a) possibly adding to said liquid sample areagent solution comprising a chaotropic substance having preferably aconcentration able to rupture hydrogen bonds; (b) filtering the liquidsample through a filter having a pore size which is small enough toprevent passage of microorganisms through the filter but large enough topermit passage of any soluble material present in the sample, wherebythe microorganisms are retained on the filter; (c) possibly passing saidreagent solution comprising a chaotropic substance through the filterhaving the retained microorganisms thereon, said chaotropic agentsolution having preferably a concentration able to rupture hydrogenbonds; and (d) passing one or several labelled reagents through thefilter, at least one of said reagents being specific for themicroorganisms to be detected.
 2. Method according to the claim 1,characterised in that the chaotropic agent solution is present at aconcentration between about 4 and about 8 M (i.e a denaturingconcentration).
 3. Method according to the claim 1 or 2, characterisedin that said liquid sample is selected from the group consisting ofsputum, blood, serum, plasma, cephalo-rachidian fluid or urine obtainedfrom an animal or human patient.
 4. Method according to any one of thepreceding claims, characterised in that said microorganisms arepathogenic bacteria or pathogenic eukaryotic parasites.
 5. Methodaccording to any one of the preceding claims, characterised in that saidmicroorganims are detected simultaneously or successively.
 6. Methodaccording to any one of the preceding claims, characterised in that saidfilter is a membrane made of nitrocellulose or nylon.
 7. Methodaccording to any one of the preceding claims, characterised in that saidchaotropic agent solution comprises an element selected from the groupconsisting of urea, guanidine, thiourea, isothiocyanate or a mixturethereof.
 8. Method according to claim 7, characterised in that theconcentration of the chaotropic agents in said chaotropic agentssolution is higher than 2M.
 9. Method according to any one of thepreceding claims, characterised in that the labelled reagents specificfor the microorganisms to be detected are antibodies, possibly coupleddirectly or indirectly to a marker, preferably selected from the groupconsisting of gold micellae, enzymes, chromophores, stained latex beads,fluorophores or a mixture thereof.
 10. Kit for the rapid detection ofone or more microorganisms present in a sample, preferably in abiological sample, comprising means and media for performing the methodaccording to any one of the preceding claims, preferably filteringmembranes of predetermined pore size to retain specific microorganisms,one or several chaotropic agents, one or several labelled reagents, oneof said reagents being specific for the microorganisms to be detected.11. Kit according to claim 10, characterised in that said chaotropicagents are present in a solution comprising an element selected from thegroup consisting of urea, guanidine, thiourea, isothiocyanate or amixture thereof, said solution being preferably at the concentrationhigher than 2M.